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5 min. Blocking Reagent: Bullet Blocking One for Western Blotting

Ready-to-use / 5 min.

Bullet Blocking One is a blocking reagent for western blotting, which exhibits excellent blocking efficiency in only 5 minutes due to the amphiphilic compound used.

  • Fast blocking in 5 min.
  • Ready to use

Comparison Data: Blocking One (our standard blocking reagent) and 5% skim milk

Blocking Reagents Bullet Blocking One Blocking One 5% Skim Milk
Blocking Time 5 min. 30 min. 60 min.
Blotting Image
Sample: 20 µg of HeLa cell extract, 5 serial two-fold dilution series
SDS-PAGE: Bullet PAGE One 5-15% (Product No. 13080) with SDS Running Buffer (Product No. 30329) at 400 V for 10 min.
Blotting: Semi-dry Blotting Buffer Solution (Product No. 30650) at 10 V for 30 min
Washing: 0.1% t-TBS (Product No. 12750)
Blocking: Refer to the figure above
1st antibody: Anti-Vimentin (C-20) (Rabbit) (Product No. SC-7557-R) diluted 1:2,000, 1 hr. at RT
2nd antibody:   Anti-Rabbit IgG-HRP (Product No. SC-2004) diluted 1:100,000, 1 hr. at RT
Detection: Chemi-Lumi One (Product No. 11644), 5 min. reaction time
Detector: LAS-3000 (High mode), 15 min. exposure time

Comparison Data: Blocking Efficiency in 5 minutes

The original Blocking One, the competitors' ready-to-use blocking reagents and the conventional blocking reagents did not show enough blocking efficiency in 5 min, while Bullet Blocking One performed well.

5 min. Blocking Time Blocking Reagents Manufacturers' Recommended Blocking Time
Bullet Blocking One   30 min.
Blocking One 60 min.
Company A
blocking reagent
30 min.
Company B
blocking reagent
60 min.
Company C
blocking reagent (protein-free)
60 min.
5% Skim Milk
in 0.05% Tween® 20-TBS
60 min.
3% BSA
in 0.05% Tween® 20-TBS
60 min.

Conditions:
PVDF membranes dot-blotted with mouse serum were washed with TBS. Blocking was performed using each reagent above. Anti-mouse IgG (Cat.#SC-2005) (1:5,000 in 0.01% t-TBS) was applied, and the membranes were washed with 0.05% t-TBS. After reaction with Chemi-Lumi One Super (Cat.#02230), detection was performed using LAS-3000 (High mode) with 90 sec. exposure time.

Use as Antibody Diluent

Diluting antibody with Bullet Blocking One resulted in less non-specific binding compared to using t-TBS as diluent.

Antibody Diluent Bullet Blocking One (Undiluted) 0.1% t-TBS
Blotting Image
Sample: 10 µg of HeLa cell extraction, 3 serial two-fold dilution series
SDS-PAGE: Bullet PAGE One 5-15% (Product No. 13080) with SDS Running Buffer (Product No. 30329) at 400 V for 12 min.
Blotting: Semi-dry Blotting Buffer Solution (Product No. 30650) at 10 V for 30 min.
Washing: 0.1% t-TBS (Product No. 12750)
Blocking: Bullet Blocking One, 5 min.
1st antibody: Anti-Cox4 (D-20) (Goat) (Product No. SC-69359) diluted 1:500, 1 hr. at RT
2nd antibody:   Anti-Goat IgG-HRP (Product No. SC-2350) diluted 1:5,000, 1 hr. at RT
Detection: Chemi-Lumi One Super (Product No. 02230), 1 min. reaction time
Detector: LAS-3000 (High mode), 10 min. exposure time

Please note;
For antibody dilution, use Bullet Blocking One undiluted, or dilute up to 20x with TBS or PBS containing 0.05 – 0.1% detergent, such as Tween 20. The appropriate dilution ratio depends on antibody conditions, such as type and concentration, pretest is required.

Comparison with 5% Skim Milk

Bullet Blocking One showed good blocking efficiency in 5 min. Although optimizations of dilution ratio to types of antibody are required beforehand, in this experiment, diluting the antibody with Bullet Blocking One resulted in the strongest signal.

Antibody Diluent Condition 1 Condition 2 Condition 3
Blotting Image
Blocking Reagents Bullet Blocking One Bullet Blocking One 5% Skim Milk
Blocking Time 5 min. 5 min. 60 min.
Antibody Diluent Bullet Blocking One
Diluted 20x
5% Skim Milk 5% Skim Milk
1st Antibody Anti-UCP1 Diluted 2,000x Room temperature, 1 hr.
2nd Antibody Anti-Rabbit IgG Diluted 2,000x Room temperature, 1 hr.

Data courtesy of Associate Professor Goto Tsuyoshi / Kim Minji, Functions of Food that Support Everyone's Health, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University

Comparison of Blocking Time and Efficiency with 5% Skim Milk

Analysis of Background Noise on Dot Blotting Data

Dot Blotting Data

Conditions:
PVDF membranes dot-blotted with mouse serum were washed with TBS. Blocking was performed using each reagent above. Anti-mouse IgG (Product No. SC-2005) (1:5,000 in 0.01% t-TBS) was applied, and the membranes were washed with 0.05% t-TBS. After reaction with Chemi-Lumi One Super (Product No. 02230), detection was performed using LAS-3000 (High mode) with 90 sec. exposure time. Data analysis was done with Multi Gauge.

Ordering Information

Product Cat.No. Storage Quantity Price(US$)  
Bullet Blocking One for Western Blotting 13779-14 4°C 200 ml 40.00 Buy
Bullet Blocking One for Western Blotting 13779-01 4°C 1 L 180.00 Buy